Curcumin composition for liver disease and preparation method of FGF21 functional protein co-assembly microparticles

Abstract

The application provides a curcumin composition-FGF21 functional protein co-assembly microparticle applied to liver diseases and a preparation method thereof, and comprises the following steps: extracting turmeric by dynamic warm soaking, obtaining turmeric filtrate after centrifugation, concentration and microfiltration; preparing high-purity curcumin composition through PS-NVP specific purification and 80% ethanol gradient elution; and preparing Curs-FGF21 co-assembly microparticles through oil-water phase assembly and centrifugation by taking a polymer as a carrier, compounding the curcumin composition with FGF21 protein. The microparticle can improve drug stability and bioavailability, realizes long-acting slow release, can regulate liver cell lipid metabolism, reduce liver lipid accumulation and inflammation, significantly improves liver damage and liver fibrosis, and has outstanding liver protection and repair effects. The process is stable and controllable, has outstanding innovation, and provides a brand-new candidate preparation research and development idea and technical scheme for the prevention and treatment of metabolic liver diseases.

Description

Technical Field

[0001] This invention relates to the fields of traditional Chinese medicine preparation and biomedical materials, and in particular to a curcumin composition-FGF21 functional protein co-assembled microparticle for liver diseases and its preparation method. Background Technology

[0002] Curcumin is a diketone compound extracted from the rhizomes of plants in the ginger family and Araceae family. It possesses various pharmacological activities, including anti-inflammatory, antioxidant, anticancer, and lipid-lowering effects, and shows broad application prospects in the pharmaceutical, food, and health product industries. However, its extraction process often involves a large amount of lipid-soluble impurities such as oils and resins, which not only affect the purity of curcumin but also increase the difficulty of subsequent purification. Furthermore, curcumin has poor stability and is easily degraded by pH, temperature, and light, limiting its efficacy. Therefore, developing specific curcumin purification methods to ensure its stability and effectiveness is crucial.

[0003] In vitro and in vivo studies have shown that curcumin can significantly improve the degree of hepatic steatosis and fibrosis by regulating fatty acid metabolism and inhibiting liver inflammation; for example, it can inhibit inflammatory signaling pathways to reduce TNF. α, IL It exerts anti-fibrotic effects by inhibiting cytokine levels in six cytokines, blocking astrocyte activation, and reducing collagen deposition. Oral curcumin shows good safety within a dose range of 0.5–8 g / day, but extremely low plasma concentrations (peak values ​​only 0.5–1.8 µmol / L) severely hinder clinical translation. Simply increasing purity cannot solve the problem of rapid in vivo metabolism; it is necessary to combine technologies such as nanodelivery, cocrystallization, or liposomes to achieve sustained release and targeted delivery, thereby improving the bioavailability of curcumin.

[0004] Fibroblast growth factor 21 (FGF21) is an endocrine factor secreted by the liver that regulates glucose and lipid metabolism, enhances insulin sensitivity, and inhibits hepatic fat accumulation and inflammatory responses. Studies have shown that exogenous FGF21 or its analogues can significantly improve metabolic liver diseases such as NAFLD, NASH, and liver fibrosis. However, FGF21 itself has a short half-life and is easily degraded by enzymes, posing pharmacokinetic limitations when used alone. Co-loading curcumin and FGF21 functional proteins into hydrophilic / hydrophobic amphiphilic self-assembled microparticles can achieve synergistic anti-inflammatory, anti-fibrotic, and metabolic regulatory effects within the same carrier, and target the liver through the microparticle shell. Curcumin reduces hepatocellular inflammation and fibrosis by inhibiting inflammatory pathways; FGF21 activates metabolic pathways to promote fatty acid oxidation and inhibit lipogenesis. The complementary targets of these two substances promise a multidimensional synergistic therapeutic strategy for liver anti-inflammatory, anti-fibrotic, and metabolic regulation. By utilizing protein-based self-assembled microparticles that protect the activity of FGF21 with a hydrophilic shell and contain curcumin in a hydrophobic core, the problems of solubility and half-life of both can be solved simultaneously.

[0005] Currently, there are no reported solutions for the comprehensive treatment of liver diseases that utilize curcumin and FGF21 to form composite microparticles through protein co-assembly to achieve multi-target synergy and precise liver delivery. Therefore, developing novel delivery systems based on curcumin-FGF21 co-assembled microparticles can overcome the pharmacokinetic limitations of single-drug therapy, providing multiple therapeutic effects including anti-inflammatory, anti-fibrotic, and metabolic regulation, and possessing liver-targeting potential, thus meeting the urgent need for efficient and low-toxicity treatment options for liver diseases. This innovative direction also provides clear technical guidance and innovative ideas for the subsequent development of products for the prevention and treatment of liver diseases. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention obtains a high-purity curcumin composition through dynamic temperature extraction combined with PS-NVP specific purification technology. Furthermore, the curcumin composition is used to construct a polymer carrier co-assembled microparticle system, achieving synergistic sustained release of the two active components. This significantly improves formulation stability and in vivo efficacy, effectively alleviating lipid metabolism disorders, liver inflammation, and liver fibrosis. It solves the technical pain points of traditional curcumin preparations, such as low bioavailability, lack of sustained release, and weak single-component intervention effects.

[0007] To achieve the above objectives, a first aspect of the present invention provides a method for preparing curcumin composition-FGF21 functional protein co-assembled microparticles, the method comprising: S1: Turmeric slices were extracted using a dynamic temperature soaking method. The resulting extract was centrifuged, concentrated, and microfiltered to obtain turmeric filtrate. S2: The curcumin filtrate was enriched, fractionated and eluted using PS-NVP specific purification technology. The eluent was collected, concentrated and freeze-dried to obtain the curcumin composition. S3: The curcumin composition and FGF21 functional protein were added to the matrix carrier and stirred and mixed. Then, a cross-linking agent was added to obtain a matrix-drug mixture, which was named the first hydrophilic phase. S4: The first hydrophilic phase was added to the liquid paraffin oil phase containing 2% polysorbate 80 and stirred to disperse, thus obtaining a water-in-oil type primary emulsion; then the water-in-oil type primary emulsion was mixed with the second hydrophilic phase to obtain a water-oil-water type double emulsion; the paraffin oil phase in the double emulsion was separated and the water layer was collected and freeze-dried to obtain curcumin composition-FGF21 functional protein co-assembled microparticles.

[0008] Further, the dynamic temperature-infusion extraction parameters for turmeric slices in step S1 are as follows: extraction speed is 100-200 r / min, extraction solvent is 50%-80% ethanol, the ratio of turmeric slices to extraction solvent is 1:10-30, the temperature-infusion is 40-60℃, the extraction time is 20-40 min per extraction, and the number of extractions is 1-3 times; the microfiltration process uses a spiral wound membrane module with a molecular weight cutoff range of 10~100 kDa.

[0009] Furthermore, in step S2, the PS-NVP specific purification preparation technology integrates a sample injection homogenizing pump, a PS-NVP purification column, and a gradient mobile phase module; the PS-NVP purification column has a specification of (120-200) mm × (50-100) mm, the particle size of the packing material in the column is 30-60 μm, and the sample loading is 5%-15% of the packing material weight; The enrichment and fractionation elution conditions are as follows: the eluent is a 20%-90% aqueous ethanol solution, the elution volume is 3-20 times the column volume, and the elution flow rate is 50-150 mL / min.

[0010] Further, in step S3, the matrix carrier is selected from at least one of the following: chitosan quaternary ammonium salt / gelatin composite system, chitosan / sodium alginate composite system, and sodium alginate / gelatin composite system; based on the dry weight of the matrix carrier, the mass ratio of the matrix carrier to the curcumin composition is 10-20:1, and the mass ratio of the curcumin composition to the FGF21 functional protein is 4-10:1; the crosslinking agent is glyoxal, and based on the volume of the matrix carrier, the volume percentage concentration of the crosslinking agent is 0.1%.

[0011] Furthermore, in step S3, the matrix carrier is a chitosan quaternary ammonium salt / gelatin composite system, wherein the mass ratio of chitosan quaternary ammonium salt to gelatin is 2:1; based on the dry weight of the matrix carrier, the mass ratio of the matrix carrier to the curcumin composition is 10-20:1, and the mass ratio of the curcumin composition to the FGF21 functional protein is 4-10:1; the crosslinking agent is glyoxal, and based on the volume of the matrix carrier, the volume percentage concentration of the crosslinking agent is 0.1%.

[0012] Further, in step S4, the second hydrophilic phase is a 1% polyvinyl alcohol solution; the volume ratio of the first hydrophilic phase, the liquid paraffin oil phase, and the second hydrophilic phase is 2-4:5-12:1-2.

[0013] The second aspect of the present invention provides a curcumin composition-FGF21 functional protein co-assembled microparticle prepared by the method of the first aspect.

[0014] Furthermore, the microparticles achieve a curcumin loading rate of 16%-20% and an encapsulation rate of not less than 95%.

[0015] Furthermore, the particle size of the microparticles is 1-2 μm, and the release rate of the curcumin composition in the microparticles in three simulated environments in vitro—healthy tissue, cancerous tissue, and human serum—meets the following requirements: not higher than 40% after 0.5 hours and not lower than 82.82% after 48 hours.

[0016] The third aspect of the present invention provides the use of the curcumin composition-FGF21 functional protein co-assembled microparticles described in the second aspect in a drug for the prevention and treatment of liver diseases.

[0017] Furthermore, the drug dosage meets the following parameters: In HepG2 cell experiments, the microparticle dosage is 2-10 μM / well based on curcumin content; in mouse models of liver disease induced by a high-fat, high-fructose, and high-cholesterol diet, the microparticle dosage is 50-200 mg / kg based on the curcumin composition content.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) High purification efficiency and clear composition of curcumin composition: This invention uses dynamic temperature soaking method (50℃, 75% ethanol) for efficient extraction, followed by 10-100KD spiral wound membrane microfiltration to remove impurities, and finally uses PS-NVP specific purification system for gradient elution. The curcumin transfer rate reaches 94.58%, the purity of the final product is ≥70.38%, and the proportion of the three main components (bisdemethoxycurcumin, demethoxycurcumin, curcumin) is clear.

[0019] (2) Co-assembled microparticles significantly improve the solubility and sustained-release properties of curcumin: Curcumin is encapsulated in a hydrophobic core, solving the problems of poor water solubility and low bioavailability. In vitro release results show that the release rate of the curcumin composition in the microparticles is no higher than 40% at 0.5h and no lower than 82.82% at 48h, exhibiting good sustained-release characteristics.

[0020] (3) Effectively protects the activity of FGF21 protein and achieves synergistic effect of two components: the hydrophilic shell encapsulates FGF21 to avoid its enzymatic inactivation; curcumin (anti-inflammatory / anti-fibrotic) and FGF21 (regulates glucose and lipid metabolism) target complement each other, achieving a multidimensional synergistic effect of anti-inflammatory-anti-fibrotic-metabolic regulation in the same microparticle.

[0021] (4) Uniform particle size and good stability, with potential for liver targeting: The particle size is distributed between 1 and 2 μm, with uniform spherical shape, and it is stable for more than a week at room temperature. The suitable particle size and the positively charged surface of chitosan quaternary ammonium salt are conducive to passive / active targeting of the liver, reducing systemic side effects.

[0022] (5) Significantly treats metabolic liver diseases with promising application prospects: It has a synergistic therapeutic effect on diseases such as non-alcoholic steatohepatitis (NASH) and liver fibrosis, overcomes the pharmacokinetic limitations of single drugs, and provides a new technical solution for the treatment of liver diseases with high efficiency and low toxicity. Attached Figure Description

[0023] Figure 1 Flowchart for the preparation of curcumin-FGF21 functional protein co-assembled microparticles.

[0024] Figure 2 The images show the purification and analysis of the curcumin composition. In the images, A is a control image of the fractionation eluent for the curcumin composition; B is a liquid phase analysis image of the curcumin composition; and C is an image of the appearance of the lyophilized sample of the curcumin composition.

[0025] Figure 3 The images show the co-assembled microparticles of curcumin and FGF21 functional protein; where A is an analysis diagram of the drug loading and encapsulation efficiency of the microparticles; B is a 10x microscope image of the microparticles; C is a particle size distribution diagram of the microparticles; and D is a diagram of the docking between curcumin and FGF21 protein molecules.

[0026] Figure 4 The study investigated the effects of Curs-FGF21 microparticles on lipid accumulation and inflammatory response in HepG2 cells. A shows an Oil Red O stained microscopic image (100x magnification); B shows quantitative Oil Red O staining; C shows triglyceride levels in each group; D shows total cholesterol levels in each group; and E, F, and G show changes in IL-1β, IL-6, and TNF-α levels in each group, respectively.

[0027] Figure 5 The effects of Curs-FGF21 microparticles on lipid accumulation and liver damage in mouse liver were investigated using the following methods: A. Body weight; B. Liver weight; C. Liver weight index; D. Serum ALT level; E. Serum AST level; F. Hepatic acetylcholine (HE) staining and Oil Red staining of liver tissue; G. Liver tissue NAS score; H. Liver triglyceride level; I. Liver total cholesterol level; J. Serum glucose level; K. Serum high-density lipoprotein cholesterol level; L. Serum low-density lipoprotein cholesterol level.

[0028] Figure 6 The effects of Curs-FGF21 microparticles on liver fibrosis and inflammatory response in mice were investigated. The results included: A. Sirius red staining; B. COL1A1 mRNA expression level in the liver; C. FN1 mRNA expression level in the liver; D. Acta2 mRNA expression level in the liver; E. Liver IL-1β level; F. Liver IL-6 level; and G. Liver TNF-α level.

[0029] Figure 7To investigate the expression of key genes involved in the regulation of bile acid metabolism in mouse liver by Curs-FGF21 microparticles: A. FXR mRNA expression level in liver; B. SHP mRNA expression level in liver; C. BSEP mRNA expression level in liver; D. MRP2 mRNA expression level in liver; E. CYP7A1 mRNA expression level in liver; F. CYP8B1 mRNA expression level in liver. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this specification, and not all embodiments. The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed herein.

[0031] The first aspect of this invention provides a method for preparing curcumin-FGF21 functional protein co-assembled microparticles, such as... Figure 1 As shown, the specific steps include: S1: Turmeric slices were extracted using a dynamic temperature soaking method. The resulting extract was centrifuged, concentrated, and microfiltered to obtain turmeric filtrate. S2: The curcumin filtrate was enriched, fractionated and eluted using PS-NVP specific purification technology. The eluent was collected, concentrated and freeze-dried to obtain the curcumin composition. S3: The curcumin composition and FGF21 functional protein were added to the matrix carrier and stirred and mixed. Then, a cross-linking agent was added to obtain a matrix-drug mixture, which was named the first hydrophilic phase. S4: The first hydrophilic phase was added to the liquid paraffin oil phase containing 2% polysorbate 80 and stirred to disperse, thus obtaining a water-in-oil type primary emulsion; then the water-in-oil type primary emulsion was mixed with the second hydrophilic phase to obtain a water-oil-water type double emulsion; the paraffin oil phase in the double emulsion was separated and the water layer was collected and freeze-dried to obtain curcumin composition-FGF21 functional protein co-assembled microparticles.

[0032] In a preferred embodiment of the present invention, in step S1, curcumin in turmeric slices is extracted using a dynamic temperature-infusion method. The extraction parameters are set as follows: extraction speed is set to 100-200 r / min, extraction solvent is 50%-80% ethanol, the ratio of turmeric slices to extraction solvent is 1:10-30 (g / ml), the infusion temperature is 40-60℃, the extraction time is 20-40 min per extraction, and the number of extractions is 1-3. The obtained extract is filtered, centrifuged and concentrated, and then microfiltered using a spiral wound membrane assembly to obtain turmeric permeate; wherein the spiral wound membrane assembly has a molecular weight cutoff range of 10-100 kDa.

[0033] In a preferred embodiment of the present invention, step S2 employs PS-NVP specific purification technology to enrich and fractionate the curcumin filtrate. The PS-NVP specific purification technology integrates a sample introduction device, a uniform flow pump, a PS-NVP purification column, and a gradient mobile phase module. The eluent is then concentrated and freeze-dried to obtain the curcumin composition. The specific process includes: The turmeric filtrate was enriched and fractionated using PS-NVP specific purification technology. The sample was injected into the turmeric permeate using a uniform flow pump with a sample loading of 5%-15% of the packing weight and an injection flow rate of 70-90 mL / min. The PS-NVP purification column had a size of (120~200) mm × (50~100) mm, and the enrichment packing material was PS-NVP polymer microspheres with a particle size of 30-60 μm. The enrichment and fractionation elution conditions were set as follows: the mobile phase was 20%-90% ethanol aqueous solution, the elution volume was 3-20 times the column volume (BV), and the elution flow rate was 50-150 mL / min.

[0034] The obtained ethanol eluent was concentrated to 100 mg / mL~150 mg / mL, freeze-dried, and the curcumin composition was obtained and stored in the dark.

[0035] In a preferred embodiment of the present invention, the matrix carrier in step S3 is selected from at least one of the following: chitosan quaternary ammonium salt / gelatin composite system, chitosan / sodium alginate composite system, and sodium alginate / gelatin composite system; more preferably, the chitosan quaternary ammonium salt / gelatin composite system is used as the matrix carrier, and the mass ratio of chitosan quaternary ammonium salt to gelatin is 2:1.

[0036] In a preferred embodiment of the present invention, in step S3, the curcumin composition ethanol solution and the FGF21 functional protein solution are added to the matrix carrier in stages. To avoid ethanol affecting the activity of the FGF21 functional protein, the curcumin composition is added preferentially until the curcumin composition in the matrix solution is completely mixed, and then the FGF21 functional protein is added. To accurately add the curcumin composition and FGF21 functional protein, the amount added is based on the dry weight of the matrix carrier, with a mass ratio of matrix carrier to curcumin composition of 10-20:1 and a mass ratio of curcumin composition to FGF21 functional protein of 4-10:1.

[0037] In a preferred embodiment of the present invention, in step S3, in order to solidify the drug mixture inside the droplets, glyoxal with a volume percentage concentration of 0.1% (based on the volume of the matrix carrier) is added to the matrix carrier of the mixed curcumin composition and FGF21 functional protein as a crosslinking agent, so that the chitosan quaternary ammonium salt / gelatin matrix completely encapsulates the drug, and a matrix and drug mixture is obtained, which is named the first hydrophilic phase.

[0038] In a preferred embodiment of the present invention, in step S4, the second hydrophilic phase is a 1% polyvinyl alcohol solution, and the volume ratio of the matrix to the drug mixture (first hydrophilic phase), the liquid paraffin oil phase, and the second hydrophilic phase is 2-4:5-12:1-2.

[0039] The second aspect of the present invention provides a curcumin composition-FGF21 functional protein co-assembled microparticle prepared by the above steps S1-S4, wherein the microparticle has a curcumin loading rate of 16%-20% and an encapsulation rate of not less than 95%; the particle size of the microparticle is 1-2 μm, and in three simulated environments in vitro—healthy tissue, cancerous tissue, and human serum—the release rate of the curcumin composition in the microparticle meets the following requirements: not more than 40% after 0.5 hours and not less than 82.82% after 48 hours.

[0040] The second aspect of this invention provides the application of curcumin composition-FGF21 functional protein co-assembled microparticles in drugs for the prevention and treatment of liver diseases. During the experiment, the drug dosage meets the following parameters: In HepG2 cell experiments, the microparticle dosage is 2~10 μM / well based on curcumin content; In mouse models of liver disease induced by a high-fat, high-fructose, and high-cholesterol diet, the microparticle dosage is 50~200 mg / kg based on curcumin composition content.

[0041] The present invention will be described in detail below through examples, but the scope of protection of the present invention is not limited thereto. In the following examples, the pharmaceuticals and reagents are all conventional commercially available products.

[0042] Example 1 This embodiment illustrates the extraction, purification, and preparation process of the curcumin composition, including: (1) Turmeric Extract 500g of turmeric slices were weighed using an electronic balance and added to the reaction vessel. Then, 10L of 75% ethanol solution was added as the extraction solvent. Dynamic temperature extraction was started at 50℃, with an extraction speed of 150 r / min and an extraction time of 20min / time. The extraction was repeated twice. After each extraction, the extract was filtered and the two filtrates were combined. The filtrate was concentrated by centrifugation and then clarified using a spiral wound membrane with a molecular weight cutoff of 10 KD to obtain the turmeric permeate.

[0043] (2) Purification preparation column loading In this embodiment of the invention, the enrichment packing material is PS-NVP polymer microspheres with a particle size of 60 μm. The PS-NVP purification column has a specification of 120×70 mm, a column volume of about 800 mL, and is loaded with about 300 g of packing material. After loading, it is washed with 90% ethanol and pure water respectively until the baseline equilibrium state is reached.

[0044] (3) Injection conditions The experimental object of this invention embodiment is the turmeric permeate extracted in step (1), the sample loading is 10% of the weight of the packing material, and the flow rate of the uniform flow pump is set to 80 mL / min.

[0045] (4) Elution conditions After the turmeric permeate was injected, it was washed with pure water for 2 column volumes (BV), followed by 3 BV of 30% ethanol solution, and finally 20 BV of 80% ethanol solution. The elution flow rate was set to 100 mL / min. During the elution process, the eluent, water wash, 30% ethanol eluent, and 80% ethanol eluent were collected.

[0046] (5) Sample collection The 80% ethanol eluent collected in step (4) was concentrated to 100 mg / mL to 150 mg / mL and then freeze-dried using a freeze dryer to obtain the curcumin composition, which was then stored in the dark.

[0047] Example 2 This embodiment illustrates the preparation process of curcumin composition-FGF21 protein co-assembled microparticles, including: First, chitosan quaternary ammonium salt / gelatin was prepared as a matrix carrier. Then, curcumin composition and FGF21 functional protein were encapsulated. Finally, the final co-assembled microparticles were formed using a W / O / W emulsification method. The hydrophilic phase was collected by centrifugation. The specific steps are as follows: (1) Preparation of chitosan quaternary ammonium salt / gelatin matrix carrier 2g of chitosan quaternary ammonium salt was weighed using an electronic balance and dissolved in 100 mL of pure water. The chitosan quaternary ammonium salt was then heated and stirred using a water bath stirrer until a uniform 2% (w / v) chitosan quaternary ammonium salt solution was formed. Subsequently, 1g of gelatin was added to the chitosan quaternary ammonium salt solution and soaked for 2 hours. After soaking, the solution was heated in a water bath at 50°C for 30 minutes with stirring during the water bath heating to obtain a uniform 2% chitosan quaternary ammonium salt / 1% gelatin matrix carrier.

[0048] (2) Preparation of matrix and drug mixture (first hydrophilic phase) B. Weigh a certain amount of curcumin composition and dissolve it in an ethanol solution to prepare a 100 mg / ml curcumin composition ethanol solution. Weigh a certain amount of FGF21 functional protein and prepare a 20 mg / ml FGF21 functional protein solution. While stirring, add 2.73 ml of the 100 mg / ml curcumin composition ethanol solution to the 2% chitosan quaternary ammonium salt / 1% gelatin matrix. After the curcumin composition in the matrix solution is completely mixed, add 2.725 ml of the 20 mg / ml FGF21 functional protein solution and continue stirring and mixing. After mixing evenly, add 0.1% (v / v) glyoxal based on the volume of the 2% chitosan quaternary ammonium salt / 1% gelatin matrix carrier to completely encapsulate the drug in the chitosan quaternary ammonium salt / gelatin matrix, thus obtaining the first hydrophilic phase of the matrix and drug mixture.

[0049] (3) Preparation of water-in-oil colostrum 100 mL of the matrix and drug mixture (first hydrophilic phase) was added dropwise to 300 mL of liquid paraffin oil containing 2% polysorbate 80 (Tween 80) using a syringe. The mixture was stirred continuously for 5 seconds at a time, with an interval of 10 seconds, and repeated 10 times to obtain a water-in-oil colostrum.

[0050] (4) Preparation of water-oil-water type double emulsion After the water-in-oil colostrum is mixed evenly, it is allowed to stand for a period of time until the water-in-oil colostrum separates into layers, with the upper layer being the oil phase. Then, 30 mL of a 1% polyvinyl alcohol (PVA) solution (the second hydrophilic phase) is added dropwise, and the mixture is stirred and mixed. After 10 minutes, stirring is stopped to obtain a water-oil-water double emulsion.

[0051] (5) Separation and drying The oil and aqueous phases in the water-oil-water complex emulsion were separated according to density. The centrifugation was performed at 4000 rpm for 10 min. The upper layer of paraffin oil hydrophobic phase in the complex emulsion was separated by a sampler. The remaining aqueous phase was collected and placed in a freeze dryer and freeze-dried at -40℃ to 37℃ to form a freeze-dried powder, which is the curcumin composition-FGF21 protein co-assembled microparticle, abbreviated as Curs-FGF21.

[0052] Analysis example 1 This analysis uses liquid chromatography to analyze the changes in total solids and curcumin purity at each stage of the preparation of the curcumin composition in Example 1. The specific details are as follows: (1) Liquid phase detection and analysis This analysis example uses Waters UPLC for liquid chromatography detection, and the detection conditions are set as follows: column: Unitary C18 (4.6×150 mm, 5µm, P / N: 11190510515). Mobile phase: A. Acetonitrile, B. 0.1% phosphoric acid solution; Samples: Curcumin and turmeric eluent; Injection volume: 10 µL; Detection wavelength: 430nm; Column temperature: 30℃; Flow rate: 1 mL / min; The elution methods are shown in Table 1: Table 1 Elution conditions (2) Calculation methods for total solids yield, transfer rate and purity The calculation methods for total solids yield, transfer rate, and purity in this analysis example are as follows: Total solids yield (%) = (total solids mass in solution / total medicinal material mass) × 100%; Curcumin purity (%) = (curcumin content in solution / total solid content in solution) × 100%; Curcumin transfer rate (%) = (Total curcumin in this step / Total curcumin in the previous step) × 100%.

[0053] Using the above calculation formula, the total solids yield and curcumin purity of each step in the preparation of the curcumin composition in Example 1 were calculated, and the results are shown in Table 2.

[0054] Table 2. Comparison of total solids yield and curcumin purity at different stages of the turmeric process. (3) Results Analysis As shown in Table 2, the curcumin purity of the turmeric extract increased by 13.11% after filtration through a 10KD spiral wound membrane, indicating that filtration removed most of the ineffective substances. Figure 2 As shown in A, after enrichment by the PS-NVP purification column, a large amount of curcumin was adsorbed on the column. The eluent, water wash and 30% ethanol eluent were transparent and the curcumin content was 0, indicating that no curcumin component flowed through. After elution with a high-ethanol solution, the purity of curcumin in the 80% ethanol eluent was found to be 70.38%, which is 33.46% higher than that of the turmeric permeate. Moreover, the curcumin transfer rate in this process was as high as 94.58%, indicating that the PS-NVP packing material can efficiently purify and enrich curcumin-like components.

[0055] like Figure 2 As shown in B, the liquid phase analysis results show that the 80% ethanol eluent mainly contains three components: bis(demethoxy)curcumin, demethoxy)curcumin, and curcumin, with contents of 35.6%, 26.6%, and 37.8%, respectively.

[0056] The separately collected 80% ethanol eluent was concentrated to 100 mg / mL~150 mg / mL, freeze-dried, and finally a water-insoluble orange-yellow powder was obtained (see...). Figure 2 The C in the text refers to the curcumin composition, which should be stored away from light and refrigerated.

[0057] In summary, the PS-NVP purification preparation technology can significantly improve the purity and purification efficiency of curcumin compositions, and the composition of the lyophilized powder is clearly defined (including bis(demethoxy)curcumin, demethoxy)curcumin and curcumin).

[0058] Analysis example 2 This analytical example illustrates the performance of the curcumin composition-FGF21 protein co-assembled microparticles (Curs-FGF21 microparticles) prepared in Example 2. The specific details are as follows: (1) Detection of Curs-FGF21 microparticle loading and embedding rate Take 1 ml of Curs-FGF21 microparticle carrier solution, add 1 ml of ethyl acetate, and stir. Separate the organic phase containing curcumin (ethyl acetate), and determine the amount of free curcumin in the microparticle solution using a UV-Vis spectrophotometer. The absorbance is set to 430 nm during the measurement. Calculate the Curs-FGF21 microparticle loading amount and embedding rate percentage using the following formula: Sample loading capacity = (total curcumin - free curcumin) / total particulate carrier × 100%; Encapsulation rate = (Total curcumin - Free curcumin) / Total curcumin × 100%; (2) Analysis of Curs-FGF21 particle size and stability After the Curs-FGF21 microparticles were prepared, they were stored at -4°C and at room temperature (in the dark) for several days. The emulsion droplets after the separation of the oil phase in step (5) of Example 2 were observed using a vertical optical microscope. The particle size was detected using a particle size analyzer, and the particle size distribution range was analyzed using MeizsMcs6.0 software (Minz Precision) to observe the stability of the microparticles.

[0059] (3) Analysis of the release rate of Curs-FGF21 microparticles in an in vitro simulated environment To evaluate drug release from microparticles, a curcumin composition was used as the monitoring target, and dialysis was performed in a 37°C water bath. Phosphate-buffered saline (PBS) media with pH values ​​of 7.4 and 5.4 were prepared to simulate healthy and cancerous tissue conditions, respectively. 5 mL of the prepared free curcumin solution and 5 mL of Curs-FGF21 microparticle solution were poured into dialysis bags (Mw cutoff 3500 g / mol), and then immersed in 15 mL of PBS media with pH values ​​of 7.4 and 5.4, respectively. At time intervals of 0, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h, 200 μL of solution was extracted and replaced with an equal volume of fresh buffer medium transferred to the sample. The curcumin content of the extracted solution was detected using liquid chromatography and calculated according to the following formula: Curcumin release rate = curcumin release / curcumin load × 100%; Here, curcumin loading refers to the amount of curcumin encapsulated, and curcumin release refers to the amount of curcumin released from the microparticles.

[0060] (4) Results Analysis from Figure 3 As shown in Figure A, the higher the amount of curcumin added, the higher the encapsulation rate and drug loading efficiency of the Curs-FGF21 microparticle carrier for curcumin. When the amount of curcumin composition added is 40 mg, the drug loading rate is 16%, and the encapsulation rate reaches 98%. When the amount of curcumin composition added exceeds 60 mg, the drug loading rate exceeds 20%, and the encapsulation rate gradually decreases from 98%. Therefore, the Curs-FGF21 microparticles can achieve a curcumin drug loading rate of 20% and an encapsulation rate as high as 95%, demonstrating good sample loading effect, high drug loading, and reduced drug loss during preparation.

[0061] Through microscopic observation (see Figure 3 (B) and particle size analysis (see B) Figure 3 As can be seen from C), the Curs-FGF21 microparticles prepared by this invention are all spherical with a golden-yellow core, and the particle size is distributed within 1~2μm. They have uniform particle size and good dispersion effect.

[0062] The microparticle solution was left at room temperature for several days, and the distribution of microparticles in the solution was observed. It was found that the microparticles were stable in morphology within one week, and the content of free curcumin in the solution remained unchanged, indicating that the prepared Curs-FGF21 microparticles have good stability.

[0063] Analysis through intermolecular interactions (see) Figure 3As shown in D), the curcumin composition (bis(demethoxycurcumin), demethoxycurcumin, and curcumin) exhibits strong binding affinity to the FGF21 protein. The release of the drug from Curs-FGF21 microparticles was monitored using the curcumin composition as the target, and the results are shown in Table 3. Table 3 shows that in three simulated environments—healthy tissue (pH=7.4), cancerous tissue (pH=5.4), and human serum—the curcumin composition showed a release rate of <40% at 0.5 h and >82.82% at 48 h, further demonstrating that the microparticles meet the sustained-release requirements for drug formulations.

[0064] Table 3 Release rate of curcumin composition in Curs-FGF21 microparticles Application Example 1 This application example illustrates the use of the Curs-FGF21 microparticles prepared in Example 2 in intervening in hepatic cell lipid metabolism and inflammatory responses. The specific details are as follows: 1. Experimental Methods (1) Cell culture Hepatocytes were cultured in DMEM-H complete medium containing 10% FBS and 1% penicillin-streptomycin, and the medium was routinely stored in a 37°C, 5% CO2 cell culture incubator. The medium was changed every 2 days during culture. When the cell confluence reached 70-80%, the cells were digested with 0.25% trypsin and passaged at a ratio of 1:2 to 1:3.

[0065] (2) Cell grouping All experiments used HepG2 liver cells from passages 5 to 60. When the cell fusion rate reached 70%-80%, the cells were digested with trypsin, the cell suspension was collected, and the cell density was adjusted to 1×10⁶ cells / year. 5 Cells were cultured at a density of 10 cells / mL in wells for 24 h. The cells were then divided into a normal control group (Control), a model group (OA, 125 μM), a microparticle group (Curs-FGF21, 4 μM), and an obeticholic acid positive control group (OCA, 5 μM).

[0066] (3) Cell modeling and drug administration After culturing cells in an incubator for 24 h, the oleic acid stock solution (50 mM) was diluted with complete culture medium to a working concentration of 125 μM. Except for the normal group, all other groups were treated with oleic acid for 24 h to induce a high-lipidemia model. Then, the appropriate concentration of the drug was prepared using complete culture medium as a solvent or diluent, sterilized through a 0.22 μm needle filter, and replaced with the oleic acid-containing culture medium. Cells were then cultured for another 24 h.

[0067] (4) Oil Red O staining to assess lipid accumulation levels in cells HepG2 cells (1×10) 5 Cells were seeded in 24-well plates with 1 mL of cell culture medium per well. After culturing for 24 h, cell models and drug administration were performed according to the aforementioned treatment method. The culture medium was removed from the cell culture plate, and the cells were washed once with PBS. The cells were then fixed with 4% paraformaldehyde for 10 min, washed twice with PBS, and then fixed and covered with 60% isopropanol for 20 s. Finally, the cells were stained with Oil Red O staining solution for 20 min, rinsed with PBS, and observed and photographed under a microscope. In addition, 500 μL of isopropanol was added to dissolve lipids in the cells, and the absorbance was measured at 520 nm.

[0068] (5) Detection of triglyceride (TG) and total cholesterol (TC) levels in cells by biochemical colorimetric method HepG2 cells (2×10) 5 Cells were seeded in 6-well plates with 2 mL of cell culture medium per well and cultured at 37°C in a 5% CO2 incubator for 24 h. Then, cell modeling and drug administration were performed according to the previously described treatment method. The old culture medium was discarded, and the cells were washed once with PBS. 150 μL of RIPA lysis buffer was added to each well to evenly cover the cells, and lysis was performed at 4°C for 25 min. Cells were then collected using a cell scraper, and the suspension was transferred to a 1.5 mL EP tube. The cells were repeatedly pipetted 60 times to ensure complete lysis, and then incubated on ice for 10 min, with repeated pipetting during this period. The cells were centrifuged at 4°C and 12000 r / min for 5 min, and the supernatant was collected. Protein concentration was determined using the BCA method, and subsequent assays for triglycerides (TG) and total cholesterol (TC) were performed according to the instructions of the respective kits.

[0069] (6) ELISA detection of inflammatory factor levels in cell culture supernatant HepG2 cells (2×10) 5 (2 mL of cell culture medium was seeded into each well of a 6-well plate. After culturing for 24 h at 37°C in a 5% CO2 cell culture incubator, the cells were used for modeling and drug administration according to the aforementioned treatment method. The cell culture supernatant was collected and centrifuged at 12,000 rpm for 10 min at 4°C. The IL-6, IL-1β, and TNF-α ELISA kits were administered according to the manufacturer's instructions.

[0070] (7) Statistical methods Data were processed using SPSS 21.0 and GraphPad Prism software 8.1. Quantitative data are expressed as mean ± standard deviation (±s). One-way ANOVA was used for multiple comparisons between groups.

[0071] 2. Experimental Results (1) Regulates lipid metabolism in liver cells from Figure 4 As shown in A, B, C, and D, compared with the Control group, the oleic acid-induced model group (OA) showed a large number of red lipid droplets in HepG2 cells, and the levels of triglycerides and total cholesterol were significantly increased (P<0.01), indicating intracellular lipid metabolism disorder and severe lipid accumulation; while after Curs-FGF21 microparticle intervention, the number of red lipid droplets in the cells was significantly reduced (P<0.01) (see...). Figure 4 (B in the text). Simultaneously, triglyceride and total cholesterol levels also decreased significantly (P<0.05) (see section B). Figure 4 (C and D in the text). The above results indicate that Curs-FGF21 microparticles can effectively improve lipid metabolism disorders in HepG2 cells.

[0072] (2) Reduce the inflammatory response of liver cells from Figure 4 As can be seen from E, F, and G, compared with the Control group, the levels of IL-1β, IL-6, and TNF-α in the culture supernatant of HepG2 cells induced by oleic acid were significantly increased (P < 0.05), indicating that the cells in the OA group underwent an inflammatory response. Compared with the OA group, both the Curs-FGF21 group and OCA significantly reduced the levels of IL-1β, IL-6, and TNF-α (P < 0.05), inhibiting the cellular inflammatory response.

[0073] Application Example 2 This application example illustrates the use of the Curs-FGF21 microparticles prepared in Example 2 in intervening in liver diseases such as hepatic steatosis, hepatic fibrosis, and hepatitis in mice. 1. Experimental Methods (1) Animal model establishment and drug administration After a week of environmental acclimatization, male C57BL / 6J mice were randomly divided into two groups: a normal control group (Control, n=9) and a model group (n=27). The normal control group was fed a standard maintenance diet, while the model group was fed a high-fat, high-fructose, and high-cholesterol diet (containing 40% fat, 20% fructose, and 2% cholesterol). After 16 weeks of continuous feeding, the model group was randomly divided into three groups according to body weight (n=9 per group): the model group (MASH), the Curs-FGF21 microparticle group (Curs-FGF21, 100 mg / kg), and the obeticholic acid group (OCA, 40 mg / kg). All three groups continued to maintain a high-fat diet, while the Curs-FGF21 group and the OCA group were administered the corresponding drug solutions by gavage, and the Control group and the MASH group were administered an equal volume of drinking water by gavage. The drug intervention lasted for 8 weeks.

[0074] (2) Sample collection After the last administration, mice in each group were fasted for more than 8 hours, anesthetized with isoflurane, and blood was collected from the eyeballs into 1.5 mL sterile centrifuge tubes. The tubes were allowed to stand at room temperature for 2 hours, centrifuged at 3000 r / min for 15 min, and the supernatant was collected for liver function and lipid-related tests. After blood collection, the liver was removed and weighed on an analytical balance. A small piece of liver tissue was taken from the edge of the largest lobe of the liver and fixed with 4% paraformaldehyde for HE, Oil Red O staining, and Sirius Red staining. The remaining liver tissue was placed in cryovials and stored at -80℃.

[0075] (3) Histopathological analysis of mouse liver tissue ① Paraffin sections of mouse liver tissue Mouse liver tissue preserved in 4% paraformaldehyde for 48 h was taken, cut into slices about 5 mm thick, and placed in an embedding cassette with running water overnight.

[0076] Dehydration treatment: The tissue was soaked in ethanol solutions of varying concentrations for dehydration in sequence, specifically: first soaked in 50% ethanol solution for 36 min, then soaked in 60% ethanol solution for 24 min, then soaked in 70% ethanol solution for 12 min, then soaked in 80% ethanol solution for 8 min, then soaked in 90% ethanol solution for 2 min, and finally soaked in anhydrous ethanol for 5 min.

[0077] Clearing treatment: The dehydrated tissue was soaked in the following solutions in sequence: first soaked in a mixture of xylene and anhydrous ethanol in a volume ratio of 1:1 for 10 min, then soaked in pure xylene (Ⅰ) for 5 min, and finally soaked in pure xylene (Ⅱ) for 5 min.

[0078] Embedding process: The transparent tissue was immersed in liquid paraffin at 60°C and kept warm for 2 hours to allow the paraffin to fully penetrate and embed the tissue.

[0079] Sectioning: Cut the embedded tissue block into thin slices with a thickness of 4 μm. Use a glass slide to take the slices out of the 37°C warm water, unfold them and attach them to the glass slide, and then store them in a -20°C refrigerator for later use.

[0080] ② H&E staining of mouse liver tissue Baking: Place the tissue sections at 60℃ and bake for 0.5~1 h to ensure that the sections adhere firmly to the glass slide.

[0081] Dewaxing: The slices after baking are dewaxed in sequence: first soaked in pure xylene (I) for 30 min, then soaked in pure xylene (II) for 20 min, and finally soaked in a mixture of xylene and anhydrous ethanol in a volume ratio of 1:1 for 5 min.

[0082] Hydration: The dewaxed sections were placed in a series of ethanol solutions for rehydration: 100% ethanol for 6 min, 90% ethanol for 2 min, 80% ethanol for 3 min, 70% ethanol for 2 min, and 50% ethanol for 2 min. Finally, they were washed with pure water.

[0083] Staining: The hydrated sections were stained in the following order: hematoxylin staining for 28 seconds, water washing, differentiation with 1% hydrochloric acid alcohol for 11 seconds, water washing, blueing with 2% sodium bicarbonate solution for 11 seconds, water washing, eosin staining for 28 seconds, water washing, and xylene clearing treatment for 2 minutes.

[0084] Mounting: The stained sections were mounted with neutral resin and observed under a microscope.

[0085] ③ NAS score of mouse liver tissue The staining results of liver tissue pathological sections from each group of mice were analyzed, and the NAS scoring system proposed by the American Association for the Study of Liver Diseases (AAS) was used to determine MASH. A NAS score of 4 or higher was diagnosed as MASH, a score less than 3 was excluded, and a score in between was considered possible. See Table 4 for detailed rules.

[0086] Table 4 Liver NAS scoring system (4) Sirius red staining of mouse liver tissue The dehydration, embedding, and sectioning methods are the same as those for the "paraffin section of mouse liver tissue" in step (3).

[0087] Dewaxing treatment: First soak in xylene (I) for 10 min, then soak in xylene (II) for 5 min, then soak in xylene (III) for 5 min; then soak in anhydrous ethanol for 2 min, 90% ethanol for 2 min, 80% ethanol for 2 min, and 70% ethanol for 2 min in sequence to completely remove paraffin and rehydrate the tissue.

[0088] Staining treatment: After dewaxing and rehydration, the sections were stained in Sirius red staining solution for 1 hour, then washed with water, stained with hematoxylin staining solution for 10 minutes, and finally washed with water to remove excess staining solution.

[0089] Clearing treatment: Immerse the stained sections in xylene (I) and xylene (II) for 5 minutes each to clear the sections.

[0090] Mounting: The transparent sections were mounted with neutral resin and observed under an optical microscope.

[0091] (5) Oil Red O staining of mouse liver tissue Embedding and sectioning: Embed the tissue blocks with OCT embedding medium. In a -20°C cryogenic chamber, cut the tissue blocks into 5-8 μm thin sections. Attach the sections to clean glass slides and allow them to air dry at room temperature for 30 minutes.

[0092] Hydration: Immerse the dried slices in distilled water 1-2 times to wash away the embedding agent.

[0093] Staining: Immerse the sections in freshly prepared Oil Red O staining solution; stain at room temperature in the dark for 15 minutes.

[0094] Differentiation and washing: Remove the sections and quickly immerse them in 60% isopropanol twice, each time for about 30 seconds; immediately rinse the sections slowly with a thin stream of running tap water for 1 minute to stop differentiation and thoroughly wash away the isopropanol.

[0095] Hematoxylin counterstaining: Immerse the sections in hematoxylin staining solution for 1-2 minutes.

[0096] Blueing process: Rinse off excess hematoxylin with tap water; immerse in 1% hydrochloric acid alcohol for 1-3 seconds to differentiate, then quickly remove; rinse again with tap water for 5-10 minutes to allow it to return to blue.

[0097] Mounting: Use filter paper to absorb excess water from the slide, add a drop of melted glycerin gelatin to the tissue, and carefully cover with a coverslip to avoid air bubbles.

[0098] Observation and analysis: After the sealing tablet has solidified, observe it under an optical microscope.

[0099] (6) The levels of glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, ALT, and AST in mouse serum were detected by biochemical colorimetric method. The levels of glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, ALT, and AST in mouse serum were detected according to the instructions of their respective biochemical reagent kits. Finally, the absorbance of the products was measured at the maximum absorption wavelength using an ELISA reader.

[0100] (7) The levels of triglycerides and total cholesterol in mouse liver tissue were detected by biochemical colorimetric method. Approximately 100 mg of liver tissue was collected and 1 mL of RIPA lysis buffer containing 1% (v / v) protease inhibitor and phosphatase inhibitor was added, along with 2 mm grinding beads. The mixture was homogenized at 4°C and 90 Hz for 120 s, followed by lysis at 4°C for 30 min. The lysate was centrifuged at 13,000 × g for 10 min at 4°C, and the supernatant was collected. The total protein concentration was determined using the BCA method, and the triglyceride and total cholesterol levels in the supernatant were detected using a microplate reader according to the kit instructions.

[0101] (8) The levels of IL-1β, IL-6 and TNF-α in mouse liver tissue were detected by enzyme-linked immunosorbent assay (ELISA). The method for extracting protein from mouse liver tissue is the same as step (7). IL-1 levels in mouse liver tissue were detected according to the respective ELISA kit instructions. 1β, IL 6. TNF The α level was determined by measuring the absorbance of the product at its maximum absorption wavelength using an enzyme-linked immunosorbent assay (ELISA) reader.

[0102] (9) The expression levels of key genes in the bile acid metabolism pathway in mouse liver tissue were detected by RT-qPCR. 0.1g of mouse liver tissue was collected and homogenized with 1 mL of lysis buffer to extract total RNA. The OD260 / 280 values ​​of the extracted RNA, as measured by a micro spectrophotometer, were between 1.9 and 2.2, indicating that the purity met the requirements. Subsequently, the RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions. Using the cDNA as a template, qPCR amplification was performed using primers synthesized by Sangon Biotech (Shanghai) (sequences shown in Table 5). The reaction program was: 95°C pre-denaturation for 60s; followed by 40 cycles of 95°C denaturation for 15s, 65°C annealing for 15s, and 72°C extension for 60s. GAPDH was used as an internal control gene, and 2... The relative expression level of the target gene mRNA was calculated using the ΔΔCt method.

[0103] Table 5 Primer Sequences (10) Statistical methods Statistical analysis was performed using GraphPad Prism 8.1 and SPSS 21.0 software. Data are expressed as mean ± standard error (SEM). One-way ANOVA was used for comparisons among multiple groups, followed by Tukey's test. A p-value < 0.05 was considered statistically significant.

[0104] 2. Experimental Results (1) Curs-FGF21 microparticles reduce lipid accumulation and liver damage in the liver. To clarify the ameliorative effect of Curs-FGF21 microparticles on MASH mice induced by a high-fat, high-fructose, and high-cholesterol diet, this invention analyzed the effects from multiple dimensions, including body weight, histology, and glucose and lipid metabolism.

[0105] The results showed that Curs-FGF21 microparticles significantly inhibited body weight gain in MASH mice starting from the fifth week of administration (P<0.01) (see [link to relevant documentation]). Figure 5 (A) Reduced liver weight and liver index (P<0.05) (see A in the original text). Figure 5 (B and C in the text); improved liver morphology and protected hepatocyte function by significantly reducing serum ALT and AST levels (P<0.05) (see B and C in the text). Figure 5 (D and E in the text).

[0106] Histological staining (HE, Oil Red) showed that Curs-FGF21 microparticles effectively reduced hepatic steatosis, inflammatory infiltration, and lipid accumulation in the liver (see...). Figure 5 (in F), reduce liver tissue NAS score (see Figure 5 (G in the range) (P<0.05).

[0107] In terms of glucose and lipid metabolism, Curs-FGF21 microparticles can significantly reduce serum glucose, total cholesterol (TC), triglycerides (TG), and LDL-C levels, increase HDL-C levels, and reduce the accumulation of TC and TG in the liver (see...). Figure 5 (H, I, J, K, L in the range) (P<0.05).

[0108] (2) Curs-FGF21 microparticles reduce liver fibrosis and liver inflammation To clarify the ameliorative effect of Curs-FGF21 microparticles on liver fibrosis and liver inflammation induced by a high-fat, high-fructose, and high-cholesterol diet in MASH mice, this study analyzed the effects from histological, fibrosis-related gene, and inflammatory factor perspectives.

[0109] Sirius red staining (see) Figure 6 Results A) showed that no obvious collagen fiber deposition was observed in the liver tissue of the Control group, while a large number of red collagen fibers were observed in the liver tissue of the MASH group, indicating liver fibrosis. After treatment with Curs-FGF21 microparticles and OCA, collagen fiber deposition in the liver tissue was significantly reduced.

[0110] Fibrosis-related gene expression analysis (see) Figure 6 (B, C, D) indicates that the mRNA expression of COL1A1, FN1, and Acta2 in the MASH group was significantly increased (P<0.05), suggesting the activation of liver fibrosis process; the Curs-FGF21 microparticle group reduced the expression of the above genes to some extent (P<0.05).

[0111] Liver inflammatory factor level detection (see) Figure 6 The results (E, F, G) showed that the levels of IL-6, IL-1β and TNF-α in the liver of the MASH group were significantly increased (P<0.05), indicating that an inflammatory response occurred in the liver; the Curs-FGF21 microparticle group could significantly reduce the levels of these pro-inflammatory factors (P<0.05).

[0112] In summary, Curs-FGF21 microparticles can alleviate liver fibrosis by inhibiting the expression of genes such as COL1A1, FN1, and Acta2, while reducing the levels of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α in the liver to alleviate liver inflammation.

[0113] (3) Curs-FGF21 microparticles regulate the expression of key genes in bile acid metabolism from Figure 7 As shown in A~F, compared with the normal group, the mRNA expression levels of FXR, SHP, BSEP, and MRP2 in the liver tissue of MASH model mice were significantly reduced (P<0.05) (see Figure 1). Figure 7 (A, B, C, D in the original text), while the mRNA expression levels of CYP7A1 and CYP8B1 were significantly increased (P<0.05) (see A, B, C, D in the original text). Figure 7 The results (E and F in the data) suggest that under GAN diet induction, the balance of bile acid metabolism in the liver of mice was disrupted, and both the synthesis and excretion pathways were disordered.

[0114] After drug intervention, the expression of each gene showed a reversal trend, with varying degrees of upregulation of FXR, SHP, BSEP, and MRP2 mRNA expression (P<0.05) (see...). Figure 7 (A, B, C, D in the original text), while the mRNA expression of CYP7A1 and CYP8B1 was inhibited to varying degrees (P<0.05) (see A, B, C, D in the original text). Figure 7 (E and F in the text). The above results indicate that Curs-FGF21 microparticles can effectively regulate the expression of key genes in bile acid metabolism, improve metabolic disorders, and thus exert a therapeutic effect on metabolism-related steatohepatitis.

[0115] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing curcumin-FGF21 functional protein co-assembled microparticles, characterized in that, The method includes: S1: Turmeric slices were extracted using a dynamic temperature soaking method. The resulting extract was centrifuged, concentrated, and microfiltered to obtain turmeric filtrate. S2: The curcumin filtrate was enriched, fractionated and eluted using PS-NVP specific purification technology. The eluent was collected, concentrated and freeze-dried to obtain the curcumin composition. S3: The curcumin composition and FGF21 functional protein were added to the matrix carrier and stirred and mixed. Then, a cross-linking agent was added to obtain a matrix-drug mixture, which was named the first hydrophilic phase. S4: The first hydrophilic phase was added to the liquid paraffin oil phase containing 2% polysorbate 80 and stirred to disperse, thus obtaining a water-in-oil type primary emulsion; then the water-in-oil type primary emulsion was mixed with the second hydrophilic phase to obtain a water-oil-water type double emulsion; the paraffin oil phase in the double emulsion was separated and the water layer was collected and freeze-dried to obtain curcumin composition-FGF21 functional protein co-assembled microparticles.

2. The method according to claim 1, characterized in that, The dynamic temperature-infusion extraction parameters for turmeric slices in step S1 are as follows: extraction speed is 100-200 r / min, extraction solvent is 50%-80% ethanol, the ratio of turmeric slices to extraction solvent is 1:10-30, the temperature-infusion is 40-60℃, the extraction time is 20-40 min per extraction, and the number of extractions is 1-3. The microfiltration process uses a spiral wound membrane module with a molecular weight cutoff range of 10-100 kDa.

3. The method according to claim 1, characterized in that, In step S2, the PS-NVP specific purification preparation technology integrates a sample injection homogenizing pump, a PS-NVP purification column, and a gradient mobile phase module; the PS-NVP purification column has a specification of (120-200) mm × (50-100) mm, the particle size of the packing material in the column is 30-60 μm, and the sample loading is 5%-15% of the packing material weight; The enrichment and fractionation elution conditions are as follows: the eluent is a 20%-90% aqueous ethanol solution, the elution volume is 3-20 times the column volume, and the elution flow rate is 50-150 mL / min.

4. The method according to claim 1, characterized in that, In step S3, the matrix carrier is selected from at least one of the following: chitosan quaternary ammonium salt / gelatin composite system, chitosan / sodium alginate composite system, and sodium alginate / gelatin composite system; based on the dry weight of the matrix carrier, the mass ratio of the matrix carrier to the curcumin composition is 10-20:1, and the mass ratio of the curcumin composition to the FGF21 functional protein is 4-10:1; the crosslinking agent is glyoxal, and based on the volume of the matrix carrier, the volume percentage concentration of the crosslinking agent is 0.1%.

5. The method according to claim 1, characterized in that, In step S4, the second hydrophilic phase is a 1% polyvinyl alcohol solution; the volume ratio of the first hydrophilic phase, the liquid paraffin oil phase, and the second hydrophilic phase is 2-4:5-12:1-2.

6. A curcumin composition-FGF21 functional protein co-assembled microparticle prepared by the method of any one of claims 1-5.

7. The curcumin composition-FGF21 functional protein co-assembled microparticles according to claim 6, characterized in that, The microparticles achieve a curcumin loading rate of 16%-20% and an encapsulation rate of not less than 95%.

8. The curcumin composition-FGF21 functional protein co-assembled microparticles according to claim 6, characterized in that, The particle size of the microparticles is 1-2 μm. In three simulated environments in vitro—healthy tissue, cancerous tissue, and human serum—the release rate of the curcumin composition in the microparticles meets the following requirements: not higher than 40% after 0.5 hours and not lower than 82.82% after 48 hours.

9. The use of the curcumin composition-FGF21 functional protein co-assembled microparticles according to any one of claims 6-8 in a medicament for the prevention and treatment of liver diseases.

10. The application according to claim 9, characterized in that, The drug dosage should meet the following parameters: In HepG2 cell experiments, the microparticle dosage, based on curcumin content, is 2-10 μM / well; in mouse models of liver disease induced by a high-fat, high-fructose, and high-cholesterol diet, the microparticle dosage, based on curcumin composition content, is 50-200 mg / kg.

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